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Regulation of RPE phagocytosis via avb5 integrin

通过 avb5 整合素调节 RPE 吞噬作用

基本信息

批准号：
8238346
负责人：
SILVIA C FINNEMANN
金额：
$ 50.03万
依托单位：
FORDHAM UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-03-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8238346
关键词：
Actins Affect Age Age related macular degeneration Aging Apical Binding Biological Assay Blindness CD47 Antigen Calpain Cell model Cells Circadian Rhythms Cytoskeleton Data Detection Disease Ensure Equilibrium Eye Focal Adhesion Kinase 1 Functional disorder Gene Mutation Goals Health Human Integrins Knockout Mice Life Ligands Ligation Link Lipofuscin Mitotic Molecular Mus Mutation Neural Retina PTK2 gene PTPN1 gene PTPN11 gene PTPN6 gene Pathway interactions Phagocytes Phagocytosis Phosphorylation Photoreceptors Process Protein Dephosphorylation Protein Tyrosine Kinase Protein Tyrosine Phosphatase Proteins Proteolysis Reactive Oxygen Species Reagent Regulation Research Project Grants Retina Retinal Retinal Degeneration Retinal Diseases Retinitis Pigmentosa Role Signal Pathway Signal Transduction Structure of retinal pigment epithelium Surface Testing Time Tissue Model age related aged human MERTK protein in vivo in vivo Model novel prevent receptor src-Family Kinases uptake

项目摘要

DESCRIPTION (provided by applicant): It is the long-term goal of this research project to identify molecular mechanisms used by the retinal pigment epithelium (RPE) to phagocytose photoreceptor outer segment fragments (POS) shed in a circadian rhythm in the mammalian eye and to understand how specific changes in these mechanisms due to aging and mutations cause retinal degeneration. Digesting several thousand POS disks every day for life, post-mitotic RPE cells are the most active phagocytes known. Abnormalities in RPE phagocytosis directly contribute to incurable human blinding diseases affecting millions, including age-related macular degeneration and retinitis pigmentosa. Identifying the contributions of the RPE to the fundamental retinal process of outer segment renewal is thus directly relevant to our understanding of human retinal disease. Recently, we developed and explored novel experimental approaches that allow for the first time to directly and quantitatively determine how genetic mutations and age-related changes alter activity of phagocytic proteins by the RPE in vivo and in culture. Shed POS require ligation of aV¿5 receptors of the RPE by their retinal ligand MFG-E8 to stimulate a cytoplasmic signaling pathway in RPE cells via focal adhesion kinase towards the tyrosine kinase receptor MerTK that is indispensable for efficient POS engulfment. Synchronized POS clearance via aV¿5 receptors is essential for long-term retinal health: lack of phagocytic rhythm in aging aV¿5 - deficient mice causes excessive accumulation of pro-oxidant RPE lipofuscin and loss of vision. In aim 1 we propose to identify the missing molecular links between aV¿5 and MerTK surface receptors and unravel the role of Src kinase in RPE phagocytosis. A novel substrate screen will identify direct Src substrates specific to POS phagocytosis and determine if Src activity is sufficient to promote MerTK activation and POS engulfment. Our preliminary data show that RPE cells following an initial burst of POS binding retain ample surface aV¿5 and MerTK receptors but fail to promote prolonged POS binding or engulfment signaling. In aims 2 and 3 we therefore propose to identify molecular mechanisms used by RPE cells to actively and specifically inhibit aV¿5 and MerTK functions following completion of diurnal POS clearance. We will test the hypothesis that RPE cells terminate POS phagocytosis by specific proteolysis of integrin associated proteins that severs anchorage of aV¿5 receptors to the actin cytoskeleton, and by stimulating specific protein tyrosine phosphatases that inactivate MerTK. Mechanisms that prevent untimely or excess RPE phagocytic activity have long been assumed to exist but to our knowledge never been studied. We propose that such inhibitory mechanisms may ensure the healthy balance of POS shedding and engulfment in the retina. We have procured appropriate in vivo models, specific activity assays and detection reagents for their identification

描述（由申请人提供）：本研究项目的长期目标是确定视网膜色素上皮（RPE）吞噬哺乳动物眼中昼夜节律脱落的光感受器外节片段（POS）的分子机制，并了解这些机制因衰老和突变而发生的具体变化如何导致视网膜变性。有丝分裂后的 RPE 细胞终生每天消化数千个 POS 盘，是已知最活跃的吞噬细胞。 RPE 吞噬功能的异常直接导致影响数百万人的无法治愈的人类致盲疾病，包括年龄相关性黄斑变性和色素性视网膜炎。因此，确定 RPE 对视网膜外节更新的基本过程的贡献与我们对人类视网膜疾病的理解直接相关。最近，我们开发和探索了新的实验方法，首次能够直接定量地确定基因突变和年龄相关的变化如何改变体内和培养中 RPE 吞噬蛋白的活性。脱落 POS 需要通过其视网膜配体 MFG-E8 连接 RPE 的 aV¿5 受体，以通过粘着斑激酶刺激 RPE 细胞中的细胞质信号传导通路至酪氨酸激酶受体 MerTK，这对于有效 POS 吞噬是必不可少的。通过 aV¿5 受体同步 POS 清除对于长期视网膜健康至关重要：老年 aV¿5 缺陷小鼠缺乏吞噬节律，导致促氧化 RPE 脂褐质过度积累和视力丧失。在目标 1 中，我们建议鉴定 aV¿5 和 MerTK 表面受体之间缺失的分子联系，并揭示 Src 激酶在 RPE 吞噬作用中的作用。新型底物筛选将识别 POS 吞噬作用特异的直接 Src 底物，并确定 Src 活性是否足以促进 MerTK 激活和 POS 吞噬。我们的初步数据表明，初次爆发 POS 结合后的 RPE 细胞保留了充足的表面 aV¿5 和 MerTK 受体，但无法促进延长 POS 结合或吞噬信号传导。因此，在目标 2 和 3 中，我们建议确定 RPE 细胞在完成昼夜 POS 清除后主动、特异性抑制 aV¿5 和 MerTK 功能的分子机制。我们将测试以下假设：RPE 细胞通过整合素相关蛋白的特异性蛋白水解（切断 aV¿5 受体对肌动蛋白细胞骨架的锚定）以及通过刺激使 MerTK 失活的特定蛋白酪氨酸磷酸酶来终止 POS 吞噬作用。长期以来，人们一直认为防止不合时宜或过度 RPE 吞噬活动的机制存在，但据我们所知，从未对其进行过研究。我们认为这种抑制机制可以确保视网膜中 POS 脱落和吞噬的健康平衡。我们采购了合适的体内模型、比活性测定和检测试剂来进行鉴定